Nano zinc oxide decorated latex drainage: A promising antibacterial material prevent retrograde infection associated with drainage

Abstract

Surgical site infections (SSI) represent a considerable burden for healthcare systems. Studies show retrograde infection of the drainage tube is an important cause of surgical site infection. To this end, Surgeons work in various ways to reduce the incidence of retrograde infections. Fast progress in nanoscience and nanotechnology is revolutionizing the field of medicine to improve the quality of life due to the myriad of applications stemming from their unique properties, including the antibacterial activity against pathogens. Herein, we investigate the antibacterial properties of a novel nanomaterial composed of nano zinc oxide-decorated latex drainages. These materials were produced by the hydrothermal method and characterized through field-emission scanning electron microscopy (FE-SEM), X-ray diffraction (XRD), and DLS (Dynamic light scattering techniques). Then inductively coupled plasma mass spectrometry (ICP-MS) measurements showed that nano zinc oxide on the surface of the latex drainages showed a gradient release process. The antimicrobial activity of nano zinc oxide -decorated latex drainage was evaluated against E. coli and Staphylococcus aureus, the main bacteriological agent in the retrograde infection associated with drainage. The results showed that slices and rods nano zinc oxide (SAR-ZnO) drainage tubes had the best antibacterial properties both in vivo and in vitro. In addition, the cell viability assay demonstrated that nano zinc oxide-decorated latex drainages exerted good biocompatibility. Therefore, SAR-ZnO drainage tubes can be a perfect nanomaterial against the retrograde infection associated with drainage.

Keywords

Nano-zinc oxide antibacterial drainage nanocomposites surgical site infection

Introduction

Surgical site infection (SSI) is one of the most serious complications after surgery, which brings great pain and economic burden to patients. The incidence of surgical area infection was approximately 3.4%.¹ In patients with cancer, the rate was as high as 9.4%. Many factors contribute to the SSI, such as diabetes mellitus, obesity, and duration of surgery. Thereinto, the placement of drainage tubes is an often overlooked but important factor for surgical site infection.^2–4 Bacteria can enter the wound retrograde through the drainage tube, causing an infection inside the wound. Currently, the main clinical methods used to prevent retrograde infection include removing drainage tubes as early as possible, increasing the use time of prophylactic antibiotics, and using anti-reflux drainage bags, which inevitably lead to problems such as inadequate drainage and overuse of antibiotics. Simone Scheithauer et al.⁵ used chlorhexidine-containing dressings for the prevention of drainage tube-related infections and achieved good results. However, wound dressings are easy to fall off in clinical practice, so our research group thought about whether we can start from drainage tubes to prevent retrograde infection?

In recent years, nanotechnology has developed rapidly and has been applied to many aspects of biomedical fields.^6–10 The widespread application of zinc oxide (ZnO) nanoparticles is a typical case. For example, due to its absorption of ultraviolet light and transparency to visible light, it becomes an excellent sunscreen.^11,12 At the same time, studies have shown that ZnO nanoparticles have anti-tumor and drug delivery effects, making them a new anticancer drug with great potential.¹³ As a new type of zinc carrier, zinc oxide nanoparticles play an important role in the treatment of diabetes and other diseases.¹⁴ The antibacterial properties of nanometer zinc oxide are the most widely used.^15–19 The main antibacterial mechanisms are: First, zinc oxide nanoparticles directly contact the cell wall, leading to the destruction of the integrity of bacterial cells;^20,21 Second, the zinc ions released are antibacterial ions, which damage the cell membrane, physiological activity, electron transport, etc., and are re-released after cell cleavage for repeated use;^22–24 Third, Zinc oxide can produce reactive oxygen radicals, which can oxidize various organic substances. Thus, inhibiting the growth of bacteria.²⁵

Common preparation methods of ZnO nanoparticles include hydrothermal method, gas-phase method, mechanical method, and sol-gel method, among which hydrothermal method is widely used due to its low experimental requirements and high production efficiency.

The purpose of this study is to combine ZnO nanoparticles with a commonly used latex drainage tube to prepare a kind of nano zinc oxide-decorated drainage tube with a good antibacterial effect.

Materials and methods method

Synthesis of nano ZnO drainage tubes

To facilitate operation, each medical latex drainage tube was cut to a size of 10 × 10 × 0.7 mm³, and the latex drainage tube was ultrasonic cleaned in deionized water to remove surface impurities for later use. Slices nano-zinc oxide (S-ZnO) drainage tubes fabrication: 50 mM Zinc nitrate hexahydrate (Zn (NO₃)₂·6H₂O), 80 mM Ammonium hydroxide (NH₃·H₂O) and 25 mM Hexamethylenetetramine (HMTA) were added to 400 mL deionized water bath at 85°C for 24 h, and ZnO solution was synthesized. A 0.2 mL solution was dropped on the surface of the latex drainage tube and dried at 55°C, which was called the S-ZnO drainage tube.

Rods-nano zinc oxide (R-ZnO) drainage fabrication: At 60°C, NaOH solution (0.03 Mol) was slowly added to Zinc acetate dihydrate (Zn (CH₃COOH)₂ 2H₂O) (0.01 Mol) methanol solution, and stirred for 2 h. The resulting solution (0.3 mL) was dropped on the surface of the drainage tube at 150°C until the solution evaporated to dryness. The samples were then placed in 500 mL deionized water with 25 mMol Zn (NO₃)₂·6H₂O and 25 mMol HMTA in a water bath at 85°C for 24 h in an R-ZnO drainage tube.

Slices and rods nano-zinc oxide (SAR-ZnO) drainage tube fabrication: The solution of slice nano zinc oxide dilutions were dropped on the surface to prepare rod nano zinc oxide drainage, than dried at 55°C.

Structural, chemical, and morphological characterization

Field-emission scanning electron microscopy (FE-SEM) was utilized to examine the surface topography of the specimens. The crystalline structure of the samples was determined by X-ray diffraction (XRD). Dynamic light scattering (DLS) was used to detect the nano-particle size of zinc oxide on the surface of the drainage tube.

Zn release and area mass alter

The quality of each sample was measured and recorded with an electronic balance of 1/1000 accuracy. After ultrasonic cleaning for 2 s, the samples were taken out and dried, and the quality of each sample was weighed again and recorded.

Take three 10 mL test tubes, add 5 mL PBS respectively, immerse the above samples control drainage tube in PBS and mark them respectively, stand for 48 h, take out the samples and immerse them in 5 mL fresh PBS for another 48 h; After that, fresh PBS water was replaced every 48 h for two consecutive weeks. The amount of Zn²⁺ in all PBS was detected by ICP-AES and recorded.

Antimicrobial test in vitro

E. coli and Staphylococcus aureus were cultured in Luria-Bertani broth at 37°C for 24 h.

Cell count: 100 μl of the bacterial solution was taken, diluted 10-6 fold, 100 μl of the bacterial solution was applied in LB AGAR medium for 12 h culture, and cell count was performed.

Antibacterial experiment: the concentration of bacteria was adjusted to 10⁷CFU/mL according to the number of cells. Then, 100 μL E coli and was put into four culture dish with 5 mL Luria-Bertani broth and co-cultured with latex drainage tube, S-ZnO drainage tube, R-ZnO drainage tube, and SAR-ZnO drainage tube for 8 h and 24 h, respectively. Staphylococcus bacteria do the same. 100 μl of each culture medium was taken out for 10⁵ dilutions, then 100 μl of the diluent was evenly applied to LB AGAR culture dishes and placed in a constant temperature incubator (37°C) for 24 h.

Antibacterial experiments after ultrasonic treatment: the same four new samples were treated with ultrasound (100 W) for 2 s. These new samples were co-cultured similarly for 48 h in a bacterial culture medium. After that, 100 μL of the co-medium was diluted at 10⁵ dilutions and took100 μL of diluent into LB AGAR culture dish and placed in a constant temperature incubator (37°C) for 24 h.

Calculation of antibacterial rate: count the bacterial colonies in AGAR medium by plate counting method, calculate and compare the antibacterial rate of each sample. The antibacterial rate of E. coli and S. aureus p = (Y−X)/Y × 100%, X was the number of colonies in the AGAR medium of the experimental group, Y was the number of colonies in the AGAR medium of the control group.

Antibacterial test in vivo

Twenty SD rats with a body weight of 350–450 were selected for drainage tube implantation. The operation was performed under strict aseptic conditions. Before surgery, the mice were anesthetized with isoflurane, followed by 5% chloral hydrate (0.5 mL/100 g) for intraperitoneal injection. After satisfactory anesthesia, the rats were skinned on the back and disinfected with iodophor, and the right middle incision of the back was taken about 1 cm. Four 5 cm long samples (control drainage tube, S-ZnO drainage tube, R-ZnO drainage tube, and SAR-ZnO drainage tube of the control group) were implanted into the incision to the muscle layer, and the drainage tube was fixed. The wound was covered with clean gauze and fixed with adhesive tape. After the operation, the mice were kept in separate cages in the same environment, allowed to move freely, and fed with commercial mouse food and water.

Two weeks after surgery, the mice were euthanized with carbon dioxide. The drainage tube was removed, and the internal section length of the drainage tube was about 0.5 cm and placed in 5 ml PBS water. 100 μl of co-culture medium was taken out for 10⁴ dilutions, 100 μl of diluent was applied in AGAR medium, and incubated at 37°C for 24 h. The antibacterial effect of each sample was compared by plate counting. The muscles and skin tissues around the drainage tube were taken and fixed in 4% neutral buffer formalin for paraffin embedding. The paraffin encapsulated blocks were cut into 5 mm serial sections, stained with conventional hematoxylin and eosin, and the tissue sections were observed under a light microscope, and the images were captured.

Biocompatibility test

Preparation of fibroblast suspension: Cells in a good growth state and logarithmic growth phase were counted;

The four drainage tubes were placed in a 24-well plate and pre-wetted. The number of cells inoculated was 4000, and the incubation Wells was set at 0 h, 12 h, 24 h, 48 h, and 72 h.

Culture in an incubator at 37°C;

At five time points, the medium containing 10%CCK8 was added to the Wells. After incubation at 37°C for 2 h, 100 μl supernatant was transferred to 96-well plates, and absorbance at 450 nm was measured with a microplate reader.

Statistical analysis

In this experiment, SPSS19.0 software was used for data analysis, so all the experiments were repeated at least three times, and the data were expressed as mean± standard deviation (mean ± SD). Comparison between groups was performed by one-way analysis of variance (ANOVA), and comparison between groups was performed by Bonferroni T-test. p < 0.05 was considered statistically significant.

Results

Morphological and structural properties

We successfully modified the surface of the drainage tube with a nano-ZnO array. Figure 1 shows Four kinds of samples of latex drainage tubes and the surface morphology of these samples under Field-emission scanning electron microscopy (FE-SEM) before ultrasonic treatment and after ultrasonic treatment. Peaks from X-ray diffraction (XRD) demonstrated the formation of three different ZnO nanoarrays on the surface of latex drainage tubes substrate, separately (Figure 2). DLS results showed that the size of S-ZnO ranges from 824 nm to 1483 nm, the size of R-ZnO ranges from 164 nm to 531 nm, and the size of SAR-ZnO ranges from 531 nm to 955 nm. (Figure 3)

Stability and Zn²⁺ release test

After ultrasonic cleaning, the mass change of each group was measured by a thousandth leveled electronic balance: S-ZnO group mass decreases 33 ± 6.98 mg/L, R-ZnO group decreases 8.33 ± 1.70 mg/L, and SAR-ZnO group decreases 34.67 ± 3.30 mg/L (Figure 4). In the Zn²⁺ release experiment, the release amount of Zn²⁺ in the first 2 days was 2.57 ± 0.12 mg/L in the S-ZnO group, 0.73 ± 0.06 mg/L in the R-ZnO group, 3.23 ± 0.25 mg/L in the SAR-ZnO group, and the release amount was significantly reduced in 0.07 ± 0.06 mg/L, 0.2 mg/L, 0.27 ± 0.06 mg/L separately (Figure 5).

Antimicrobial test in vitro

By co-culture with S. aureus and E. coli, we measured the antibacterial activity of each group of nano-ZnO drainage tubes at different periods. As shown in Figure 6, the antibacterial efficiency of S-ZnO and SAR-ZnO drainage tubes was higher (97.47 ± 0.61% and 98.10 ± 0.32%) in a short time (8 h). When the incubation time increased to 24 h, the antibacterial efficiency of R-ZnO significantly increased (18.37 ± 2.47% to 76.27 ± 3.98%). After ultrasonic cleaning, the antibacterial efficiency of the S-ZnO drainage tube decreased significantly (14.27 ± 3.98%, 16.27 ± 5.18%), while the antibacterial efficiency of R-ZnO and SAR-ZnO had no obvious change (from 76.07 ± 4.57%, 77.37 ± 4.33% to 81.90 ± 2.36%, 78.30 ± 9.13%).

Antimicrobial test in vivo

In the animal test, mice were implanted with normal drainage tubes and three kinds of nano-ZnO drainage tubes (Figure 7(a)). The co-culture results of drainage tubes after 2 weeks showed that the number of bacteria in the co-culture medium of nano-ZnO drainage tubes was significantly less than that in the control group (Figure 7(a)). The antibacterial rate of these nano zinc oxide drainage tubes was measured by comparing them with the control group. S-ZnO group was 90.93 ± 1.43%. R-ZnO group was 85.37 ± 2.85%. SAR-ZnO group was 90.67 ± 2.20%. (Figure 7(b)) At the same time, the muscle and skin tissue sections around the internal segment of the drainage tube showed that the number of inflammatory cells in the section containing the zinc oxide group was significantly lower than that in the control group (Figure 8).

Biocompatibility test

In this study, fibroblasts (L-929 cells) were used to detect the biocompatibility of nano-ZnO drainage tubes in each group. The growth condition after co-culture with fibroblasts for 12 h, 24 h, 48 h, and 72 h showed that there was no significant difference in cell survival rate (p > 0.05) compared with the control group at each time point which means this material possess good biocompatibility (Figure 9).

Discussion

Drainage tube placement is an important risk factor for surgical site infection. And surgery site infections have been reported to complicate 10 to 20% of surgeries.²⁶. Domestic and foreign scholars have carried out a large number of studies on catheter-associated infection. Among them, Lang et al.²⁷ used an antimicrobial-impregnated shunt catheter to make the drainage have antibacterial properties, which reduced the infection rate of CSF from 9% to 0.6%.²⁷ But the use of antibiotics brings with it the problem of drug resistance. In addition, Lajcak et al.²⁸ used silver-impregnated catheters to reduce the EVD infection rate significantly,²⁸ but the toxicity of silver is a problem that cannot be ignored. In addition, several studies have encapsulated antibiotics and inorganic antibacterial ions in hydrogels and then coated them on the surface of catheters for antibacterial activities which achieved good results.^29,30 But the stability of hydrogel on the surface of the drainage tube was poor, and it was easy to be removed. The purpose of this study was to investigate a nano zinc oxide-coated drainage tube with a gradient antibacterial property that met the clinical needs exactly.

Stability and gradient release capability

Zinc oxide as an attractive material has aroused great interest worldwide, especially due to the implementation of the synthesis of nanoscale particles. But there are little studies of zinc oxide drainage tubes to be reported. We prepared three nano-zinc oxide coatings using zinc nitrate as a zinc source. The coating is modified on the surface of the drainage tube by hydrothermal method. Studies have shown that the antibacterial properties of nano zinc oxide are determined by the nanometer level, zinc oxide concentration, and surface-to-volume ratio.³¹ DLS tests show that our zinc oxide is nanoscale (Figure 3). And by changing the concentration of zinc acetate, the load of the R-ZnO on the surface of the latex tube can be adjusted. In this way, we changed the concentration and surface-to-volume ratio of ZnO nanoparticles. R-ZnO coatings have better stability under ultrasonic treatment than S-ZnO (Figure 4). The specific reason we have not studied it in more depth, we hypothesized that the S-ZnO could not stick to the uneven surface of the drainage tube, while the R-ZnO could still completely fill in the pit of the drainage tube. Vanessa Valdiglesias et al.³² ʼs experiment showed that when zinc ion concentration reached 25 mg/L or above, it would produce cytotoxicity.³² The results of our zinc ion release experiment were much lower than this concentration, ensuring the safety of the drainage tube (Figure 5). The gradient release capacity of the SAR-ZnO drainage tube is the guarantee of gradient antibacterial.

Antibacterial effect in vitro and in vivo

The antibacterial properties of zinc oxide nanoparticles are of great significance in the medical field.^33–35 Nano zinc oxide has significant growth inhibition on a wide range of bacterial types.^36,37 During postoperative drainage, the exposed drainage tubes are bound to adhere to various bacteria. The nano-zinc oxide drainage tubes we prepared exhibit strong antibacterial activity against the most common infectious pathogens in the clinic (S. aureus and E. coli) in vivo and in vitro (Figure 6 and Figure 7). In the three kinds of nanosized zinc oxide drainage tubes prepared by us, the zinc ions in the slices nanosized zinc oxide can be released quickly in a short period of time, which makes it show a significant antibacterial effect in a short period of time. However, due to the insufficient stability of its binding on the surface of the drainage tube, the slices of nano zinc oxide were largely removed after ultrasonic treatment, and its antibacterial effect was significantly decreased. The rod-shaped nano zinc oxide has good ultrasonic stability and can release zinc ions at low concentrations continuously, which has a continuous bacteriostatic effect. And the characteristics of SAR nano zinc oxide with two groups of nano zinc oxide and its gradient release characteristics of zinc ions to form the gradient antibacterial effect, more in line with the need of clinical, in the short term after surgery quickly kill the bacteria around the drainage tube, bacteriostatic, a long time our zinc ion release experiment lasted for 2 weeks there is still a low concentration of zinc ions, This indicates that our material can sustain antibacterial activity for at least 2 weeks. For a more in-depth analysis of antimicrobial, we took the muscles and skin around the drainage tube for HE staining. In the absence of nano zinc oxide as an antibacterial agent, bacterial stimulation caused the accumulation of inflammatory cells in the skin and muscle tissue (Figure 8). To prepare biomaterials that can be used clinically, it is essential to detect the biocompatibility of materials. We conducted toxicity tests with L929 fibroblasts and showed that all three nano-zinc oxide drainage tubes were biocompatible (Figure 9).

Conclusion

The SAR-ZnO drainage tube showed good antibacterial activity against S. aureus and E. coli and had a gradient release function, which could be a promising antibacterial material prevent retrograde infection associated with drainage.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by National Natural Science Foundation of China (grant no. 82060403).

ORCID iD

Li Tao

Cheng Xigao

Appendix

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Nano zinc oxide decorated latex drainage: A promising antibacterial material prevent retrograde infection associated with drainage

Abstract

Keywords

Introduction

Materials and methods method

Synthesis of nano ZnO drainage tubes

Structural, chemical, and morphological characterization

Zn release and area mass alter

Antimicrobial test in vitro

Antibacterial test in vivo

Biocompatibility test

Statistical analysis

Results

Morphological and structural properties

Stability and Zn2+ release test

Antimicrobial test in vitro

Antimicrobial test in vivo

Biocompatibility test

Discussion

Stability and gradient release capability

Antibacterial effect in vitro and in vivo

Conclusion

Footnotes

Declaration of Conflicting Interests

Funding

ORCID iD

Appendix

References

Stability and Zn²⁺ release test